Rozália Pusztai

Genotypic analysis of two hypervariable human cytomegalovirus genes

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times. J. Med. Virol. 80:1615–1623, 2008.

Are Granulocytes the Main Effector Cells of Natural Cytotoxicity in Chickens

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.

Effect of ultraviolet irradiation and heating on the interferon-inducing capacity of human adenoviruses.

Summary Heating adenovirus types 8 and 12 for 2 to 3 min. at 56° simultaneously reduced both interferon-inducting capacity and infectivity. In contrast, virus inactivated by u.v.-irradiation effectively stimulated interferon production in chick cells. Chick cells infected with adenovirus type 12 and incubated at 25°, 35° or 40° produced about the same amount of interferon but the kinetics of interferon production differed, i.e. at the higher temperatures interferon formation commenced earlier. The possibility that the penton antigen may be responsible for interferon induction by human adenoviruses in chick cells is discussed.

Chemoprevention and inhibition of P-glycoprotein in cancer cells by Chinese medicinal herbs.

Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P‐glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate‐early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro‐organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells. Copyright

Induction of Interferon in Chick Cells by Adenoviruses of Different Origin

The human adenoviruses were previously found to be effective inducers of interferon in chick cells both in vitro and in vivo (Beladi & Pusztai, 1967; Pusztai et al. 1969a). The titres of interferon obtained in chick fibroblast cells are similar to those of interferon induced by Semliki Forest and Sindbis viruses under the same conditions. An avian adenovirus (GAL) also induced interferon in chick embryo fibroblast cells (Bakay, 1969). Human adenoviruses are still infective after treatment with trypsin but are not able to stimulate interferon synthesis (Beladi & Pusztai, 1967). These results prompted us to study the effect of trypsin on the ability of other adenoviruses to induce interferon in chick cells. The present paper describes experiments in which canine hepatitis virus, bovine adenovirus type 2, simian adenovirus types 15 and 17, and GAL virus were compared with the human adenovirus type 12 as inducers of interferon in chick cells.

Opposite effects of serotonin and interferon-α on the membrane potential and function of human natural killer cells

SummaryIn an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-α (IFN-α) partially eliminates this effect. Because natural IFN-α might contain factors other than IFN, we repeated these experiments with recombinant human interferon-α (rhIFN-α) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-α on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-α applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-α hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-α antagonizes this ability via its hyperpolarizing activity.

Characterization of adenovirus type 12 tumour antigen produced in chick fibroblasts.

Tumor (T) antigen was characterized in non-permissive chick fibroblasts and permissive HEp-2 cells infected with Ad12 either in the presence or in the absence of cytosine arabinoside. Antiserum against T antigen specifically immunoprecipitated two polypeptides of apparent mol. wt. 50 000 and 11 000.

Rescue of Heat-inactivated Adenovirus Types 1 and 6 by Ultraviolet-irradiated Adenovirus Type 8

Summary Heating human adenovirus types 1 and 6 at 56° reduced infectivity very rapidly. A treatment for 10 to 15 min. usually completely destroyed the infectivity. However, the rescue of heated virus occurred when HEp-2 cells were inoculated simultaneously with heat-inactivated type 1 or 6 and u.v.-irradiated type 8. Rescue was dependent upon the length of heat treatment, as viruses heated at 56° for more than 20 min. could not be rescued. On the other hand, the rescue of heat-inactivated type 6 was related to the u.v. dose employed, i.e. type 8 unirradiated or irradiated for 20 min. was not suitable for rescue. The possible mechanism of rescue observed with heat-inactivated adenovirus types 1 and 6 by u.v.-irradiated type 8 is discussed.

Amyloid-beta interactions with ABC transporters and resistance modifiers

Background/Aim: Failure of cancer chemotherapy caused by multidrug resistance (MDR) of tumor cells is mediated by ABC transporters that reduce the uptake of cytotoxic agents. Similar transporters are responsible for amyloid clearance in nerve cells in Alzheimers disease (AD). The aim of this study was to compare the biological effects of amyloid complexes of some known ABC transporter inhibitors e.g. disiloxanes. One of the most active fragments of the pathological “endogen” substrate responsible for AD was investigated in the presence of amyloid-beta fragment on the reversal of multidrug resistance and apoptosis induction on multidrug-resistant tumor cells in model experiments. Materials and Methods: The efflux pump activity of the cells treated with amyloid-beta complexes was studied by Rhodamin-123 accumulation. Apoptosis induction was measured by staining of treated cells by Annexin-V and propidium iodine. The fluorescent activity FL-1 and FL-2 of the cells was measured and analyzed on a PARTEC FACScan instrument. Results: The resistance modifiers: disiloxanes and memantine complexed with amyloid-beta 1-42 reduced the activity of ABC transporter in MDR tumor cells. Early apoptosis was moderately increased by amyloid-beta complexes. Late apoptosis and the number of total viable cells were not changed. Conclusion: Amyloid-beta and its complexes inactivate the efflux pump of tumor cells resulting in accumulation of amyloid. It is supposed that reduced membrane transport can explain the lower incidence of cancer in AD.

The role of interferon in the adenovirus-induced augmentation of granulocyte-mediated cytotoxicity in chicken.

The effects of human adenoviruses on the granulocyte-mediated natural cytotoxicity of chicken leukocytes were investigated. A significant, but transient augmentation of granulocyte cytotoxicity was observed 24 h after virus injection, followed by a relatively long period of its suppression. A good correlation was found between the augmented cytotoxicity and interferon induction. The interferon-inducing capacity of adenovirus type 6 and type 12 in vitro similarly ran parallel with their ability to stimulate granulocyte-mediated cytotoxicity. An adenovirus-induced elevation of cytotoxicity was not observed when IFN production was inhibited by pretreatment of the leukocytes with monoclonal antibody specific for bursal cells and monocytes. In addition, anti-IFN antibody abrogated the stimulation of cytotoxicity as well. During the in vitro experiments in which granulocyte-specific monoclonal antibody was applied, evidence was found that the effector cell activity is associated with the granulocytes. These results suggest that both the in vitro and the in vivo adenovirus-induced augmentation of granulocyte-mediated cytotoxicity is due to the IFN-inducing capacity of the virus. In chickens, the rapid augmentation of the granulocyte cytotoxicity may be important in the acute stage of infection, increasing the resistance to the virus in question and also to bacterial infections.